Genotoxicity analysis

ABSTRACT

Genotoxicity testing can be carried out using a genetic hybrid cell line, for example, a CHO cell line which contains human chromosome 11. This exemplified hybrid cell line expresses human CD59 on the cell surface, and the human CD59 gene serves as a test for mutagenic agents. The hybrid cell line is grown in the presence of a test compound, and the loss of cell surface CD59 is followed using a fluorescent-labeled antibody specific for human CD59 and flow cytometry to monitor the presence or absence of labeled antibody on particular cells. Absence of the labeled antibody on the surface of the cells is indicative of a mutation in the CD59 gene such that either no CD59 protein is made or there has been a mutation which results in the loss of the antibody binding site. A test compound which causes CD59 loss is deemed to be genotoxic (i.e., mutagenic). The mutations can be point mutations, deletions, inversions, insertions, or frameshifts. The sensitivity of the assay is improved when the cells are first panned with antibody specific for the cell surface marker to remove spontaneous mutants prior to challenge with the test compound. Alternatively, or in addition, an antibiotic resistance marker is incorporated onto the same chromosome as the cell surface marker, and an antibiotic selection step precedes the challenge with the potential genotoxic composition or condition.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.60/378,422, filed May 6, 2002.

ACKNOWLEDGMENT OF FEDERAL RESEARCH SUPPORT

This invention was made, at least in part, with funding from theNational Institutes of Health (Grant No. NIH SBIR 1 R43 CA91566-01).Accordingly, the United States Government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

The field of this invention is genotoxicity testing in mammalian cells,in particular as it relates to measuring the occurrence and/or frequencyof mutant phenotypes in a hybrid cell using immunological analysiscoupled with flow cytometry or other analytical techniques.

There is a need in the art for simple, rapid and economical assaymethods for identifying compositions with negative aspects including,but not limited to, the ability to cause mutations or other geneticdamage in mammalian cells, especially where those assay methods arefast, economical and sensitive. Such assay methods improve toxicitytesting and improve the safety of materials for use in or near mammals,including humans.

SUMMARY OF THE INVENTION

This present invention provides methods for identifying genotoxic agentsby measuring loss of function in hybrid cells in which a cell surfaceprotein is expressed via a gene carried by a chromosome heterologous tothe cell (in the present specific examples). The loss of function can bedetermined by lack of binding of a detectable ligand specific to a cellsurface protein, for example, a labeled antibody binding to the cellsurface protein or a labeled second antibody specific for the antibodybound to the cell surface protein, especially a fluorescently labeledantibody or second antibody, using flow cytometry. However, other meansof identifying loss of a particular function carried by the heterologouschromosome can be employed, provided that it is possible to quantitateboth positive and negative cells so as to determine the frequency withwhich the function is lost. The hybrid cell can be derived from twodifferent species of animal; desirably the hybrid cell is anonhuman-human hybrid which maintains at least one human chromosome,from which at least one detectable protein, preferably a cell surfaceprotein, is expressed. As specifically exemplified, the assay system isa flow cytometry-based mammalian cell mutation assay system using(hybrid) Chinese hamster ovary (CHO) cells which contain a humanchromosome that encodes proteins not encoded by naturally occurring CHOcells, and the absence of which is indicative of loss of function of inthe respective encoding gene. This assay system allows the measurementof genotoxicity in mammalian cells (as required of pharmaceutical andchemical companies by the Food and Drug Administration (FDA) andEnvironmental Protection Agency (EPA) in a more rapid, cheaper and moreinformative scheme than can be achieved with the tests now employed,reducing the time required for mutant detection from days (as is nowrequired for all assays with mammalian cells) to hours. Thehuman-hamster hybrid CHO A_(L) and A_(N) cells carry human chromosome11, which encodes the CD44, CD56, CD59 and CD98 cell surface proteins.The A_(N) cells also carry the hygromycin resistance determinant on thesame human chromosome (11) as the noted cell surface marker. When thehybrid cells are cultured prior to and/or during challenge with thechallenge compound or condition, the background of cells which havespontaneously lost the relevant human chromosome or the function of thedetectable marker, e.g. cell surface protein, is reduced.

Flow cytometry is used to measure the presence or absence of aprotein(s) encoded by a human chromosome stably incorporated into aChinese hamster cell by binding of fluorescent-labeled monoclonalantibodies to the protein(s). Mutations in the gene(s) resulting in aloss of antibody binding site (or in the loss of the entire protein) areindicated by the loss of a fluorescent signal.

In the present methods the hybrid cells are cultured in the presence ofa test compound (or test condition), the test compound or condition isremoved, and the cells are allowed to divide several times before thepresence or absence of the target function is determined. When thefrequency of loss of function cells in the population is greater inthose subjected to the test compound or test condition than in cellscultured in the absence of the test compound or test condition, the testcompound or condition is deemed to be genotoxic.

Also within the scope of the present invention is an improved method forgenotoxicity testing wherein, prior to challenge with the test compound,the cells are treated to remove those cells which have already lost theability to express the cell surface marker which is measured in theassay. As specifically exemplified, the cells are panned to remove thosewhich lost the ability to express the CD59 cell surface marker. Thecells are trypsinized and contacted with CD59-specific antibody, andthen contacted with a solid support (e.g., a culture dish) which hasbeen previously coated with a second antibody which specifically bindsthe CD59-specific antibody. This pretreatment improves the sensitivityof the assay in that the background of CD59⁻ cells is reduced by fromabout 10-fold to about 200-fold or more, depending on the spontaneousloss of the detectable function. Further improvement is achieved whenpanning and antibiotic selection are combined prior to challenge of thehybrid cells with the test composition or condition.

DETAILED DESCRIPTION OF THE INVENTION

A genotoxic agent is one that can interact by binding covalently withgenetic material. Genotoxic agents can be either direct or indirectagents. Broadly speaking, genotoxic agents include both mutagenic andclastogenic agents. In the present context, a compound, composition orcondition is genotoxic when it causes mutations in at least one celltype in which it is tested or prevents expression of a target gene.

A mutagen is a chemical or physical agent that produces base-pairsubstitutions or small insertions or deletions of one or more base pairsin genetic material. As broadly used herein, mutations include pointmutations, deletions, insertions, and chromosomal rearrangements andresult in loss of function (gene expression or antigenic determinant).

A clastogen is a compound, condition or environmental agent that cancause one of two types of structural changes. A clastogen can causebreaks in chromosomes that result in the gain, loss, or rearrangementsof chromosomal segments. A clastogen can also cause sister chromatidexchanges during DNA replication”. In the context of the presentinvention, there is a loss of function (expression of a target gene orelimination within an expressed target gene of an antigenicdeterminant).

Hybrid cell lines are those in which the genotype is primarily of onespecies, e.g., a nonhuman cell such as hamster, but there is at leastone chromosome of a different species (heterologous chromosome)maintained in that cell line in a stable fashion. As specificallyexemplified herein, a hamster cell line (CHO, Chinese hamster ovary)contains human chromosome 11 (CHO A_(L)). The CHO A_(N) cell line isderived from the A_(L) line by modification to contain a hygromycinresistance determinant on human chromosome 11, so that the presence ofthe human chromosome can be selected. Selection for the presence of thehuman chromosome helps eliminate false positives in the genotoxicityassay. The heterologous chromosome directs the expression of at leastone protein on the cell surface, and that protein is antigenicallydistinct from proteins made by the cell in the absence of thatheterologous chromosome.

A test compound (or composition) is one for which information concerningits ability to cause mutations is sought. The compound is tested forgenotoxicity by culturing the hybrid cells and detecting the loss of theprotein (or an antigenic determinant thereof) whose expression isdirected by the heterologous chromosome.

A test condition is one which is analyzed for the ability to causemutation in a hybrid cell line as described herein. A test condition canbe, but not limited to, irradiation with ultraviolet light, visiblelight, radio waves, x rays, gamma rays, shortwave, electromagnetic ormicrowave irradiation, for example, or it can be high or lowtemperature, among others.

The methods of the present invention are important in that they allowthe testing for genotoxicity of chemical and physical agents using amammalian cell system. Manufacturers of drugs and chemicals are requiredto test them for genotoxicity using a variety of assays, includingmammalian cells. These methods can be used for the genotoxicity testing,and the improved economics and shorter times can save the drug andchemical companies significant amounts of money.

The assay methods of the present invention are more sensitive thanalternative mammalian cell assays. They are also more rapid and muchless expensive to use than alternative assays. It allows reduction inthe time and cost of testing drugs and other agents, which could be veryimportant for large drug and chemical manufacturers.

The A_(L) hybrid was derived almost 30 years ago by fusing a CHO cellwith a normal human fibroblast (T. T. Puck, P. Wuthier, C. Jones, and F.T. Kao. 1971. Lethal antigens as genetic markers for study of humanlinkage groups. Proc. Natl. Acad. Sci. USA 68, 3102-3106). Subcloneswere selected that stably retained a single human chromosome 11. Thishuman chromosome 11 (and all other human chromosomes 11) wassubsequently shown to encode a series of surface antigens whose mappositions on the chromosome are known (C. A. Waldren. 1983. Mutationalanalysis in cultured human-hamster hybrid cells, In: Chemical Mutagens:Principles and Methods for Their Detection, Vol. 8, (F. J. de Serres.,Ed.), pp. 235-260, Plenum Publ Corp., New York; C. Waldren, L. Correll,M. A. Sognier, and T. T. Puck. 1986. Measurement of low levels of x-raymutagenesis in relation to human disease. Proc. Natl. Acad. Sci. USA 83,4839-4843; C. A. Waldren and C. Jones, 1981. Chromosome loss and damageas measured by biological markers, In: Health Risk Analysis: Proceedingsof the Third Life Sciences Symposium, (C. R. Richmond, P. J. Walsh, andE. D. Copenhaver., Eds.), pp. 333-343, The Franklin Press, Philadelphia,Pa.). Since only one or a few genes located at the very tip of the shortarm of chromosome 11 are required for survival of the hybrid, mutationsranging from a point mutation to loss or rearrangement of chromosomalfragments of greater than 160 Mbp on chromosome 11 can be detected (S.M. Kraemer, D. B. Vannais, A. Kronenberg, A. Ueno, and C. A. Waldren.2001. Gamma-Ray Mutagenesis Studies in a New Human-Hamster Hybrid,A(L)CD59(+/−), which has Two Human Chromosomes 11 but is Hemizygous forthe CD59 Gene. Radiat. Res. 156, 10-19). The standard assay for mutationusing these cells is based on complement-mediated cytotoxicity. Cellsare labeled with antibodies against specific surface antigens (usuallyCD59) and then treated with rabbit complement. Cells which express thesurface antigen(s) are killed by the complement, but cells which aremutated and do not express the surface antigen(s) survive. This hasallowed for sensitive quantification of the mutagenic activity ofstandard “point” mutagens, e.g. ethyl-methane sulfonate (EMS), N-methylN-nitrosoguanidine (MN NG) and UV, as well as clastogens like mitomycinC and ionizing radiations (C. A. Waldren, C. A. 1983, supra; C. Waldren,C. 1986; C. Waldren, C. Jones, and T. T. Puck. 19790. Measurement ofmutagenesis in mammalian cells. Proc. Natl. Acad. Sci. USA 76,1358-1362; N. Matsukura, J. Willey, M. Miyashita, B. Taffe, D. Hoffmann,C. Waldren, T. T. Puck, and C. C. Harris. 1991. Detection of directmutagenicity of cigarette smoke condensate in mammalian cells.Carcinogenesis 12, 685-689; S. M. McGuinness, M. L. Shibuya, A. M. Ueno,D. B. Vannais, and C. A. Waldren. 1995. Mutant quantity and quality inmammalian cells (AL) exposed to cesium-137 gamma radiation: effect ofcaffeine. Radiat. Res. 142, 247-255; T. K. Hei L.-J. Wu, S.-X. Liu, D.Vannais, C. Waldren, and G. Randers-Pehrson. 1997. Mutagenic effects ofa single and an exact number of alpha particles in mammalian cells.Proc. Natl. Acad. Sci. USA 94, 3765-3769).

We have also shown that such non-genotoxic carcinogens as asbestos andarsenic are, in fact detectable as quite strong mutagens, producingmainly chromosomal mutations in A_(L) cells (T. K. Hei, S. X. Liu, andC. Waldren, Mutagenicity of arsenic in mammalian cells: role of reactiveoxygen species. 1998. Proc. Natl. Acad. Sci. USA 95, 8103-8107; T. K.Hei, C. Q. Piao, Z. Y. He, D. Vannais, and C. A. Waldren. 1992.Chrysotile fiber is a strong mutagen in mammalian cells. Cancer Res. 52,6305-6309).

The CD59 gene maps on the short arm at 11p13.5; other genes of interestsuch those encoding CD98 (also known as SLC3A2) is located on the longarm at 11q13 (FIG. 1). The CD59 gene has been cloned (D. Vannais, M.White, M. McGraw, A. Davies, A. Wilson, T. Hei, and C. Waldren. 1998.Intragenic mutations in gene MCI=CD59 can now be analyzed inhuman-hamster hybrid AL cells. Radiat. Res. 106). Its exact size isknown and PCR primers have been made which can be used to defineintragenic mutations (S. M. Kraemer et al. 2001. supra). The CD56 (alsoknown as NCAM1) gene is located at position 11q23.1 on chromosome 11.The gene located at 11p15.5 is an essential gene in the hybrid cellline, making the loss of human chromosome a lethal event. Furthermore, anew cell line (CHO A_(N)) has been generated; this cell line has thehygromycin gene stable incorporated in the long arm of chromosome 11.Spontaneous loss of chromosome 11 can be selected against by addingneomycin to the cultures to reduce the level of background mutants (C. AWaldren, B. Failed, M. Braden, R. D. Parker, and D. Vannais. 1992. Theuse of human repetitive DNA to target selectable markers into only thehuman chromosome of a human/hamster hybrid cell line (AL). Somat. CellMol. Genet 18, 417-422).

We have developed a flow cytometric assay based on CHO A_(L) cells (orCHO A_(N) cells) that allows simultaneous detection of both point andchromosomal mutations and is much more rapid and less expensive than thetraditional A_(L) assay based on complement-mediated cytotoxicity. Theassay is similar to the A_(L) assay discussed above except that thepresence or absence of the surface antigens such as CD44, CD59, CD56 orCD98 is measured by binding of specific monoclonal antibodies.Monoclonal antibodies specific for these markers are commerciallyavailable from Ancell Corporation, Bayport, Minn.; Research DiagnosticsInc., Flanders, N.J.; Biomedia, Foster City, Calif.; Sigma Chemical Co.,St. Louis, Mo., among others. We have demonstrated that the flowcytometry based method is highly sensitive and linear, and it canreadily detect mutations induced by ionizing radiation and chemicalagents such as MNNG. There are several advantages of this method overthe cytotoxicity-based A_(L) assay and the mouse lymphoma assay (MLA).First, the flow cytometry assay does not depend on colony growth, so the7-10 day period required for colonies to form in the traditional assaysis eliminated. Only a few hours are required to carry out the antibodylabeling procedures and the flow cytometry analysis. Second, it is muchless expensive because it does not require labor-intensive cell culturefor colony growth and counting of colonies. Also it does not requireexpensive and unreliable rabbit serum complement. Third, the fullydeveloped assay will be able to distinguish between small and largemutations by measuring the presence or absence of two or more antigenssimultaneously. Fourth, the A_(L) cells are extremely robust and easy tohandle with a generation time of about 12 hr, so the expression periodrequired before analysis of mutations is relatively short, desirably 7to 12 days, advantageously 9 days. This method is also improved over aprior art method which relies on magnetic separation of expressing andnon-expressing cells.

Testing of drugs by pharmaceutical and chemical companies is amulti-billion dollar enterprise which includes both in-house testing andtesting by independent companies. We have identified at least 10commercial companies that use the MLA test (an alternative mammaliancell mutation assay system). Typical costs cited by one company rangefrom $2000-4000 for a preliminary screening to $25,000 for a fullfledged GLP (Good Laboratory Practices) assay suitable for submission tothe United States FDA. The turn-around time for a full-fledged assay isabout 8 weeks. Further development and validation of the flow cytometrybased assay could cut the time in half for these assays and reduce thecost by at least 50%. Thus, the commercial potential for a more rapid,less expensive and more sensitive assay is enormous.

An example of the separation between positive cells containing the CD59antigen (A_(L)) and negative cells (parental strain which does notcontain human chromosome 11) is shown in FIG. 2. The peaks are separatedby over two orders of magnitude.

By using careful handling procedures and gating the cells on lightscatter and time of flight, we have achieved measurements of 0.03% ofnegatives within the positive population (FIG. 4) when regions were setto include >97% of negative cells (FIG. 4). This is close to thebackground level of mutants usually observed within the population ofpositive cells.

To test the accuracy of measuring a small mutant population, we did anumber of mixing experiments in which various fractions of negativecells were mixed in with positive cells. Starting with an initialmixture of 2% negatives, serial two-fold dilutions were made to aminimum of 0.125% negatives. These mixed samples were then stained withantibodies against CD59 and analyzed. As shown in FIG. 5, flow cytometrymeasurements of the percent of mixed cells is highly linear whencompared to the actual mixed percentage. The slope is 0.97 with anR-squared value of 0.998 and an intercept of 0.02 percent. These resultsdemonstrate that flow cytometry has the intrinsic sensitivity andlinearity to be useful for measuring mutant fractions induced by varioustreatments.

These calibration results are convincing that flow cytometry has thesensitivity needed to measure mutant yields induced in cells bydifferent agents. In order to actually measure mutants instead of justmixed cell populations, we irradiated populations of A_(L) cells withdoses from 0 to 4 Gy, then grew the cells for 9 days with subculturingas needed to allow the induced mutants to regain normal growth and tolose the expression of CD59 on the surface of the cells. Threeindependent samples were irradiated and subcultured for each dose. Thesamples were then processed for antibody staining and flow cytometryanalysis, using commercially available antibody and well knowntechniques. Two independent experiments have been done with very similarresults. The results shown in FIG. 6 represent one experiment with 3independent samples for each dose point. There is a linear dose responsefor mutant yield as measured by flow cytometry. The mutant yield at 0dose is somewhat high compared to historical controls in the standardcomplement-mediated assay and to other results for unirradiated controlcells using flow cytometry. Without wishing to be bound by anyparticular theory, this is believed due, at least in part, to the factthat these cells were in culture for several months and the backgroundlevel of mutants was increasing. These experiments are being repeatedusing cells which have been panned to reduce the background mutationfrequency to determine whether a lower yield at 0 dose can be obtained.Results shown in FIG. 5 indicate that we can measure a background levelof mutations of only about 0.02%. The slope of the curve is quitesimilar to published results using the complement-mediated cytotoxicityassay.

Because the separation of the positive and negative cells is so great(see FIGS. 3 and 4), the assay can be fine tuned for greatersensitivity. We have successfully measured 0.03% negative cells in abackground of positive cells (for CD59) when the regions were set toinclude >97% of the negative cells.

Flow cytometry measurements are highly linear for mixtures of positiveand negative cells when compared to the calculated percentages ofpositive and negative cells. The slope is 0.97 with an R-squared vale of0.998 and an intercept of 0.2% (FIG. 5).

There is a linear dose-response curve for mutant yield as measured byflow cytometry after radiation of the hybrid cells (FIG. 6). Themeasured background mutant level in this experiment was about 0.1%. Theassay for chemically induced mutations is very sensitive, measuring asignificant increase in mutant yield at concentrations of 0.1 μg/ml MNNG(FIG. 7). These results demonstrate that the flow cytometry mutationassay is robust in the sense that it is sensitive to mutations inducedby both radiation and alkylating agents. Thus, it has been demonstratedthat the methods of the present invention are applicable to clastogenicand other mutagenic agents.

We have also measured mutant yield at the CD59 locus induced by MNNG(FIG. 7). CHO AL cells were treated with various concentrations of MNNGfor 16 hr at 37° C. The mutagenized populations were then grown up for10 days, stained with antibody against CD59 and analyzed by flowcytometry as described. The results shown are based on 2 separateanalyses of the same experiment. These results clearly show an increasein mutations with increasing concentration of MNNG. They also indicatethat the assay is very sensitive, measuring a significant increase inmutant yield at concentrations of 0.1 μg/ml of MNNG. These results alsoshow that the flow cytometry mutation assay is robust in the sense thatit is sensitive to mutations induced by both radiation and alkylatingagents.

The results described herein above have been improved by panning thehybrid cells prior to use in genotoxicity testing studies by panning toremove those cells which, prior to challenge with a potential genotoxicagent, have lost the ability to express CD59. The cells are trypsinized,contacted with a commercially available CD59-specific mouse antibody andthe bound via the CD59-specific mouse antibody to a surface coated withantibody specific for mouse antibody. This pretreatment panning lowersthe background of CD59⁻ cells and increases the sensitivity of the assayfrom about 10-fold to 200-fold or greater, depending on the spontaneousloss of CD59 expression.

Culture of the hybrid cells prior to challenge with the test compound ortest condition in the presence of an antibiotic, where a resistancedetermination is carried on the same heterologous chromosome as theexpression product to be detected, serves to improve the sensitivity ofthe genotoxicity testing methods by reducing the background of cellswhich have spontaneously lost the heterologous chromosome. Thisantibiotic selection step can be used with or without the panning step.Where these two are combined, the results are improved significantly (atleast 2-fold) over use of either selection alone.

Monoclonal or polyclonal antibodies, preferably monoclonal, specificallyreacting with a protein of interest can be made by methods well known inthe art. See, e.g., Harlow and Lane (1988) Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratories; Goding (1986) MonoclonalAntibodies: Principles and Practice, 2d ed., Academic Press, New York;and Ausubel et al. (1993) Current Protocols in Molecular Biology, WilleyInterscience/Greene Publishing, New York, N.Y. Also, recombinantimmunoglobulins may be produced by methods known in the art, includingbut not limited to the methods described in U.S. Pat. No. 4,816,567.Monoclonal antibodies with affinities of 10⁸ M⁻¹, preferably 10⁹ to 10¹⁰or more are preferred.

Antibodies specific for particular proteins are useful, for example, asprobes for screening for loss of a functional gene encoding theparticular protein, in the context of the present invention. Desirably,the antibodies are labeled by joining, either covalently ornoncovalently, a substance which provides a detectable signal. Suitablelabels include but are not limited to radionuclides, enzymes,substrates, cofactors, inhibitors, fluorescent agents, chemiluminescentagents, magnetic particles and the like. United States patentsdescribing the use of such labels include but are not limited to U.S.Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;4,275,149; and 4,366,241. Fluorescent agents are especially useful inapplications using fluorescence activated cell sorting.

The descriptions provided herein are for illustrative purposes, and arenot intended to limit the scope of the invention as claimed. Anyvariations in the exemplified methods which occur to the skilled artisanare intended to fall within the scope of the present invention.

All references cited in the present application are incorporated intheir entirety herein by reference to the extent not inconsistentherewith.

Materials and Methods

Cell Culture

CHO A_(L) cells were cultured in F12 medium at 37° C. at 5% CO₂ in anincubator. Parental (CHO) cells without human chromosome 11 were used asa negative control. Cells were passaged every three days to avoidconfluence. To reduce background mutations, cells were either panned byusing a CD59-specific antibody or stained and sorted for CD59 positivecells as described below. When A_(N) cells are used, they are desirablycultured in medium containing 800 μg/ml neomycin before challenge withthe test compound or test condition to select against cells which havespontaneously lost human chromosome 11. The antibiotic selection can beused instead of panning or in addition to panning.

Antibody Staining

Cells were stained using 50 μl and phycoerythrin-labeled monoclonalantibody specific for human CD59 per 1×10⁶ cells.

Flow Cytometry

Cells were analyzed using a Coulter EPICS V cell sorter at 488 nm with a515 SP and 575 LP filters. Cellular debris was removed by gating onForward Scatter vs. Side Scatter. A total of 50,000 cells were collectedper sample. Cells which expressed CD59 were brightly stained, whereasmutants lacking reactivity with the CD59-specific antibody were dim.Gates were set so that 97% of the negative control parental (CHO) cellswere counted.

Calibration

To ensure test sensitivity, CHO A_(L) cells were mixed with parental CHOcells at concentrations ranging from 0.0125% to 0.5%. The mixed sampleswere then stained with the labeled CD59-specific antibody and analyzedusing flow cytometry. FIG. 5 shows typical results for assaycalibration.

Chemical Induced Mutations

CHO A_(L) cells were plated and 3 hr later MNNG was added. After 3 hrincubation, the medium was aspirated, the cells were washed with sterilephosphate buffered saline, and fresh F12 medium was added to the flasks.Cells were then grown for 7-12 days, (desirably about 9 days) andanalyzed by antibody binding and flow cytometry.

Radiation Induced Mutations

CHO A_(L) cells were plated in T75 flasks and then irradiated to reach amaximum dose of 80% toxicity. After a survival curve was developed, thecells were then irradiated at doses from 0 to 4 Gy, cultured for 7 to 12days (desirably about 9 days), contacted with the labeled CD59-specificantibody and analyzed by flow cytometry as described above.

Panning

Panning is used to significantly reduce background marker loss in theA_(L) or A_(N) cells or other hybrid cells. For A_(L) or A_(N) cells,non-tissue (non-coated) culture plates, 100 mm, i.e. those notpre-coated, are incubated with goat anti-mouse IgM antibody in PBS 10ml, 20 μg/ml, for 2 hours at room temperature to allow the IgM to bindto the plastic. The plates are then washed 3 times with PBS to removeexcess, nonadhered IgM, and then they are filled with 1% fetal bovineserum (FBS) in phosphate buffered saline (PBS) (FBS/PBS) (5 ml) andstored at 4° C. until use.

Approximately 4×10⁶ A_(L) or A_(N) cells are trypsinized, harvested in2% FBS/PBS, collected by centrifugation and resuspended in 0.2%Anti-CD59/2% FBS/PBS at a cell concentration of about 2×10⁶ per ml toallow binding of the Anti-CD59 antibody to cells which express themarker. After 30 min incubation with antibody at 4° C., the cells arerinsed with 2% FBS/PBS 3 times and then diluted in 2% FBS in PBS to give2.5×10⁶ in 5 ml in a single plate. FBS/PBS is removed from the treatedplates and then 5 ml cell suspension is added to the plates. The cellsare incubated for 2 hours at 4° C., then the PBS is aspirated, removingfloating cells. The plates are gently rinsed 3 times with 5 ml cold 2%FBS/PBS to remove unattached CD59⁻ cells (negatives). Complete medium isthen added to the plates and pipetted forcefully several times to removethe CD59⁺ cells from the plates. The cells are then collected, passagedtwice and frozen for use. At this point, the cells are very low inbackground CD59⁻ cells and are ready for use in genotoxicity testingstudies. Where a different cell surface marker is to be monitored, theappropriate antibody is used in the selection prior to contacting withthe composition or condition being tested for genotoxicity, and then thesame antibody is used to measure genotoxicity (mutagenicity and/orclastogenic activity).

1. A method for genotoxicity testing, said method comprising the stepsof (a) culturing a hybrid cell line derived from a first species and asecond species, wherein said hybrid cell line stably maintains at leastone chromosome of the first species and wherein said cell line expressesat least one cell surface protein derived from the first species, in thepresence of a test compound or a test condition for from about four toabout twenty cell divisions; (b) optionally subsequently culturing saidcells in the absence of the test compound or test condition; (c)contacting the cells cultured in step (a) or step (b) with a detectableligand specific for the first species cell surface protein; (d)subjecting the cells contacted with detectable ligand in step (c) to asorting process to quantitate cells to which the labeled antibody hasnot bound and quantitating the cells to which the labeled antibody hasbound; and (e) identifying the test compound or test condition as agenotoxic agent when there is a greater number of cells to whichantibody has not bound after culture with the test compound or conditionthan there is when the cells have been cultured in the absence of a testcompound or genotoxic compound without exposure to said composition orsaid condition.
 2. The method of claim 1, wherein the detectable ligandcomprises at least one antibody specific for said cell surface protein.3. The method of claims 1 or 2, wherein the hybrid cell line is ahuman-non-human animal hybrid cell line.
 4. The method of claim 3,wherein the hybrid cell line is a human-hamster hybrid cell line.
 5. Themethod of claim 4, wherein the human-hamster hybrid cell line is a CHOAL cell line.
 6. The method of any of claim 1, wherein said hybrid cellline expresses at least one antibiotic resistance gene, wherein saidantibiotic resistance gene and said first species cell surface proteingene resides on the at least one first species chromosome, and whereinprior to step (a), said cell line is cultured in the presence of theantibiotic to which the at least one antibiotic resistance gene confersresistance.
 7. The method of claim 4, wherein the human hamster hybridcell line is a CHO AN cell line.
 8. The method of any of claim 1,wherein the labeled antibody is a fluorescent-labeled antibody.
 9. Themethod of any of claim 3, wherein the cell surface protein is a humanprotein selected from the group consisting of CD44, CD56, a CD59 andCD98.
 10. The method of claim 8, wherein the cells are sorted byfluorescence activated cell sorting.
 11. The method of any of claim 1,wherein prior to the step (a), said method comprises the step ofremoving cells which do not express the cell surface protein.
 12. Themethod of claim 10, wherein the step of removing comprises contactingthe hybrid cells with a first antibody specific for the cell surfaceprotein and then contacting with a second antibody specific for saidfirst antibody, wherein said second antibody is bound to a solidsupport.
 13. The method of claim 10, wherein prior to step (a), thehybrid cells are cultured in the presence of an antibiotic to whichresistance is conferred by an antibiotic resistance gene linked to thecell surface protein gene in an amount and for a time sufficient to killcells which have lost ability to express the antibiotic resistance gene.